Yeast 14‐3‐3 proteins

Hemert, Martijn J. van; Heusden, G. Paul H. van; Steensma, H. Yde

doi:10.1002/yea.739

Cited by 49 publications

(43 citation statements)

References 49 publications

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“…Clearly, this simple FP assay is applicable to 14-3-3 proteins in general, very likely including yeast and plant 14-3-3 proteins. 34,35 To refine the 14-3-3 FP assay, we tested a range of different concentrations of the fluorescent Raf probe for 14-3-3 binding (0.5, 1, 2, and 5 nM) ( Fig. 2A).…”

Section: An Hts Assay For 14-3-3 Proteinsmentioning

confidence: 99%

Monitoring 14-3-3 Protein Interactions with a Homogeneous Fluorescence Polarization Assay

Masters

Khuri

et al. 2006

SLAS Discovery

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The 14-3-3 proteins mediate phosphorylation-dependent protein-protein interactions. Through binding to numerous client proteins, 14-3-3 controls a wide range of physiological processes and has been implicated in a variety of diseases, including cancer and neurodegenerative disorders. To better understand the structure and function of 14-3-3 proteins and to develop small-molecule modulators of 14-3-3 proteins for physiological studies and potential therapeutic interventions, the authors have designed and optimized a highly sensitive fluorescence polarization (FP)–based 14-3-3 assay. Using the interaction of 14-3-3 with a fluorescently labeled phosphopeptide from Raf-1 as amodel system, they have achieved a simple 1-step “mixand-measure” method for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used tomonitor the binding of 14-3-3with a variety of client proteins. The 14-3-3 FP assay is highly stable and has achieved a robust performance in a 384-well format with a demonstrated signal-to-noise ratio greater than 10 and a Z• factor greater than 0.7. Because of its simplicity and high sensitivity, this assay is generally applicable to studying 14-3-3/client-protein interactions and especially valuable for high-throughput screening of 14-3-3 modulators.

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Section: An Hts Assay For 14-3-3 Proteinsmentioning

confidence: 99%

Monitoring 14-3-3 Protein Interactions with a Homogeneous Fluorescence Polarization Assay

Masters

Khuri

et al. 2006

SLAS Discovery

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“…14-3-3 proteins are necessary elements in the regulation of a variety of biological systems. For example, 14-3-3 proteins participate in regulation of cell cycle arrest in response to DNA damage, cell cycle timing, and intracellular signaling in response to stress and mating pheromone in yeast (van Hemert et al, 2001), photoreceptor development and learning in Drosophila (Skoulakis and Davis, 1998), cellular response to stress and survival factors in mammals, and the Ras/Raf signaling pathway in various organisms (Fu et al, 2000;Tzivion et al, 2001;Tzivion and Avruch, 2002). Nearly 100 proteins have been reported to associate with 14-3-3 proteins in vivo.…”

Section: Introductionmentioning

confidence: 99%

Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding

Shen

Godlewski

Bronisz

et al. 2003

MBoC

107

117

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14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other

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“…A wide variety of regulatory functions are attributed to the 14-3-3 proteins, which in many cases are due to their ability to bind phosphoserines (Muslin et al, 1996). While seven isoforms have been identified in mammalian cells, only two 14-3-3 proteins are present in Saccharomyces cerevisiae: BMH1, BMH2 (Gelperin et al, 1995;van Hemert et al, 2001;van Heusden et al, 1992van Heusden et al, , 1995 and Sz. pombe: RAD24, RAD25 (Ford et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

The Candida albicans 14‐3‐3 gene, BMH1, is essential for growth

Cognetti

Davis

Sturtevant

2001

Yeast

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The 14-3-3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades. In Saccharomyces cerevisiae, the 14-3-3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development. The Bmh proteins function in the cAMPdependent RAS/MAPK and rapamycin-sensitive signalling cascades. Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions. Strains deleted of both BMH1 and BMH2 are either non-viable or demonstrate sensitivity to environmental stresses. In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable. The 14-3-3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT-PCR. Unlike other yeasts, C. albicans has only one 14-3-3 gene (BMH1). It was not possible to construct double knockouts by routine methods. These results suggested that the C. albicans BMH1 gene is essential. The essentiality of C. albicans BMH1 was confirmed by a PCR disruption technique. The C. albicans bmh1D/BMH1 heterozygotes exhibit growth and morphogenetic defects. Therefore, the BMH1 gene in C. albicans (Accession No. AF038154) is an excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis.

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Yeast 14‐3‐3 proteins

Cited by 49 publications

References 49 publications

Monitoring 14-3-3 Protein Interactions with a Homogeneous Fluorescence Polarization Assay

Monitoring 14-3-3 Protein Interactions with a Homogeneous Fluorescence Polarization Assay

Significance of 14-3-3 Self-Dimerization for Phosphorylation-dependent Target Binding

The Candida albicans 14‐3‐3 gene, BMH1, is essential for growth

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